IOSR Journal of Biotechnology and Biochemistry (IOSR-JBB) ISSN: 2455-264X, Volume 3, Issue 4 (Jul. – Aug. 2017), PP 19-24 www.iosrjournals.org Cleavage of obestatin by DPP-IV and inhibition of incretin effect on MIN6 cells Pooja Jaiswal1, Hamendra Singh Parmar1, Aseem Kumar Anshu1, Nikita Chordiya1 , Rakesh Patel2, Shreya Paliwal3 1(School of Biotechnology, Devi Ahilya University, Takshashila Campus, khandwa Road, Indore-452001, M.P., India.) 2(Shree Rewa Gurjar college, Sanawad-451111, dist. khargone M.P., India) 3(Department of Biosciences, SPU, Anand-388120, Gujrat, India) Corresponding Author: Pooja Jaiswal Abstract: Obestatin is a poorly characterized peptide secreted from gastrointestinal tract (GIT) and pancreatic islet. It is a 23-amino acid amidated peptide hormone formed through posttranscriptional processing of ghrelin gene. Some studies suggest that obestatin plays an important role in gluco-regulation.These features of obestatin are similar to glucagon-like peptide-1 (GLP-1), which is also synthesized and secreted from GIT and formed through the posttranslational processing of proglucagon gene.Active form of GLP-1 potentiates insulin secretion mainly in presence of food or glucose stimuli (incretin effect), but it is inactivated by the cleavage of dipeptidyl peptidase-IV (DPP- IV) enzyme. Obestatin was evaluated on MIN6 mouse pancreatic β-cells for incretin effect and survival. Cleavage and inactivation of obestatin by DPP-IV was confirmed by mass spectrometric analysis. Comparisons were made with standard GLP-1.In response to the incubation with external obestatin, endogenous secretion of obestatin was diminished, DPP-IV enzyme was found to be inhibitory on incretin effect. Mass spectrometric analysis of the incubated mixture of obestatin and DPP-IV revealed cleavage of obestatin .XTT assay and media concentration of DNA revealed positive influence of obestatin on MIN6 cells survival or proliferation. We conclude that obestatin exert incretin effect and positively influence survival of pancreatic cells similar to GLP-1.Obestatin was also found to be cleaved by DPP-IV enzyme at amidated C-terminal. Keywords: DPP-IV, Ghrelin, GIT, GLP-1, Obestatin. ----------------------------------------------------------------------------------------------------------------------------- ---------- Date of Submission: 17-07-2017 Date of acceptance: 05-09-2017 ----------------------------------------------------------------------------------------------------------------------------- ---------- I. Introduction Gluocagon-like peptide-1 (GLP-1) is one of the several products of posttranslational processing of proglucagon gene transcript in intestinal L-cells of gastrointestinal tract (GIT)[1]. This category of peptides are known as incretins, because they potentiate insulin secretion in response to food or glucose stimuli; known as incretin effect. Some other poorly characterized peptides also exert similar functions and known as glucose dependent insulinotropic peptides (GIPs) [1-3]. GLP-1 and GIPs are very important peptides to treat diabetes due to their pleiotropic effects such as stimulation of insulin secretion, diminish glucagon secretion, delayed gastric emptying, reduced hepatic glucose output and increased pancreatic β-cell survival [4]. However, cleavage of GLP- 1 through dipeptidyl peptidase-IV(DPP-IV) enzyme converted it into inactive form. DPP-IV enzyme is a subset of oligopeptidase encoded by CD26. It is widely distributed in various tissues. This enzyme cleaves off N-terminal amino acids and inactivates number of biologically active peptides including GLP-1 and GIPs. This enzyme specifically cleaves off dipeptides from protein having alanine or proline at position 2 of their N-terminal [5]. Similar to GLP-1, obestatin is also a 23-amino acid amidated peptide hormone, identified as a product of ghrelin gene (GHRL). The m-RNA of GHRL gene has four exons and through post transcriptional modifications it encoded five products of similar structures. Further, posttranslational cleavage of one product C-ghrelin (unacylated) is presumed to form obestatin in vivo [6].Biological functions of obestatin are still poorly understood but it is thought to be anorectic peptide which decreases food intake. However number of other functions including promotion of insulin secretion, survival of β-cells and pancreatic islets, decrease in weight gain, gastric emptying and jejuna motility being suggested, but mechanisms are not well understood [6-8]. Some reports also suggested that obestatin functions through G-protein coupled receptor (GPR39), which is also debatable and receptor for obestatin remains unknown [8-10].After careful review of the functions, receptor studies and amino acid sequence of human obestatin, we hypothesized that obestatin may exert incretin effect and may be cleaved by DPP-IV at its C-terminal because this is amidated and having alanine at position 2. Therefore, in present study, we addressed two major questions whether obestatin functions similar to GLP-1 and whether DPP-IV cleaves off obestatin into its inactive form. DOI: 10.9790/264X-03041924 www.iosrjournals.org 19 | Page Cleavage of obestatin by DPP-IV and inhibition of incretin effect on MIN6 cells II. Materials and methods 2.1 Chemicals Human obestatin, Gly-pro-p-nitroanilide, Dipeptidyl peptidase-IV (DPP-IV), GLP1RA (GLP-1 non peptide receptor agonist) and standard GLP-1 were purchased from Sigma-aldrich.MIN6 cells were gifted by Dr. Vasudevan Sheshadri, Scientist E, NCCS, Pune, India. Media DMEM was purchased from Hi-media Pvt. Ltd, India. Insulin and obestatin estimations were done using ELISA kits from IRI, USA. XTT kit was purchased from Hi-Media Pvt., Ltd. India. 2.2Methods 2.2.1Studies on MIN6 cells and mass spectrometric analysis MIN6 cells were cultured in DMEM initially for 5 days. Then three different concentrations (10nM, 5nM and 01 nM) of GLP-1 standard and obestatin were used in preliminary experiment. Out of these concentrations 1 nM concentration of obestatin was found suitable for insulin secretion from MIN6 cells. Therefore, all the experiments conducted at 1nM concentration of these two peptides. In each setup 20 µl of GLP-1 or obestatin was used in 200 µl of media. The incubation time for each experiment was set for 48 hrs. DPP-IV concentration was 0.05 U/ ml and 10 µl in each well it was added and incubated for 48 hrs. Media was containing 25 mM glucose [10]. Insulin and obestatin estimations in media samples were conducted by ELISA kits. Estimation of XTT was carried out using kit method. XTT was done post experimental incubation of 48 hours of cells with XTT substrate by following the protocol provided by manufacturer. DNA estimation was done using spectrophotometric analysis at 260 and 280 nm. Absorbance at 320 nm was also used to subtract from the absorbance of 260 nm to base line turbidometric errors. After calculation of DNA purity quantitation was done. Mass spectral data were obtained by recording the positive mode electro spray ionization (ESI) on a Brukermicr OTOF-Q II mass spectrometer[12]. In silico, molecular docking study was conducted using AutoDock4.0, where as to model protein structures, modeller 9.0 was used. To visualize these structures PyMol was used. These all tools are freely available online. It is noteworthy that to predict the natural way of interactions of GLP-1 and obestatin with GLP- 1R (GLP-1 receptor) and DPP-IV free or blind docking was considered, as reported elsewhere[11, 13-15]. III. Results 3.1. Effect of obestatin and GLP-1 on insulin secretion, concentrations of obestatin, XTT and DNA in media from MIN6 cells (Table 1) In this study a profound increase was observed in insulin secretion in response to administration of obestatin and standard GLP-1. The increase in insulin concentration was approximately three folds greater than control values in both the test peptides. However, in presence of external GLP-1 and obestatin into the media the overall concentration of obestatin was decreased. On the parameter of cell survival, an increase was observed in the absorbance of XTT in both GLP-1 and obestatin treated groups. In obestatin treated group, no significant change was observed in DNA concentration in media, while significant decrease was observed in GLP-1 treated group. 3.2. Effect of obestatin and GLP-1 on insulin secretion, obestatin, XTT and DNA in media from MIN6 cells in presence of DPP-IV (0.05U/ml) (Table 2) In this study, presence of DPP-IV, inhibited insulin secretion from MIN 6 cells. The most pronounced effect was observed in DPP-IV control where more than 50% decrease was observed. However, a significant increase was observed in response to obestatin and GLP-1 treated groups. No significant change was found in XTT values of DPP-IV group as compared to control. However, almost four fold increase was observed in XTT values of GLP-1 treated group, whereas three folds higher in obestatin treated group. In case of DNA concentration, it was found to be decreased only in GLP-1 treated group. 3.3 Mass spectrometric analysis of DPP-IV and obestatin (Fig 1) Mass spectrometric analysis revealed the release of a dipeptide containing alanine-leucine from obestatin, in response to incubation of obestatin along with DPP-IV enzyme. 4. Comparative study using in silico molecular docking of GLP-1 and obestatin with DPP-IV and GLP- 1Recpetor (Fig 2 and 3) We observed that both GLP-1 and obestatin interact and bind with the active cavity of DPP-IV and GLP-1 receptor; however the strength of binding was higher in case of GLP-1. DOI: 10.9790/264X-03041924 www.iosrjournals.org 20 | Page Cleavage of obestatin by DPP-IV and inhibition of incretin effect on MIN6 cells Table 1: Effects of obestatin and GLP-1 on insulin, obestatin, XTT and DNA concentration in media from MIN6 cells Groups Insulin (µU/ml) Obestatin (pg/ml) XTT(Abs) DNA(µg/ml) Control 10.52 ± 0.420 8.77 ± 0.175 0.216 ± 0.013 43.41 ± 0.546 GLP-1 a a c b 31.08 ± 2.40 6.31 ± 0.186 2.682 ± 1.062 32.086 ± 3.17 Obestatin a a c c 31.02 ± 1.160 5.40 ±0.429 2.008 ± 0.198 42.79 ± 2.039 a b c Data are means ±S.E.M.(n=3); ,P<0.001; ,P<0.01and ,P<0.05whencomparedwith the values of control.